One-Step 2-Minute Test To Detect Typhoid-Specific Antibodies Based on Particle Separation in Tubes

Abstract

Typhoid fever is caused by Salmonella typhi. Detection of anti-S. typhi antibodies in the patient is a useful diagnostic aid. Among the various methods developed over the years for this purpose, the Widal test, based on bacterial agglutination, has remained the most widely used, even though it is neither specific nor sensitive. Its popularity stems from the fact that it is simple to use and inexpensive. We describe a new test which also uses a simple one-step procedure but is more rapid and accurate than the Widal. The new test (TUBEX) detects anti-Salmonella O9 (both immunoglobulin M [IgM] and IgG) antibodies in patients by inhibiting the binding between an anti-O9 IgM monoclonal antibody (MAb) conjugated to colored latex particles and S. typhilipopolysaccharide (LPS) conjugated to magnetic latex particles. The reactants are mixed in a specially designed microtube for 2 min, and the result is read based on the resultant color of the supernatant following forced sedimentation of the magnetic beads. In the absence of inhibitory antibodies, there is a color change (from blue to red) due to cosedimentation of the indicator particles with the magnetic particles, whereas if these antibodies are present, they prevent such a change to a degree dependent on their concentration. Preliminary examination of TUBEX using the anti-O9 MAb and irrelevant MAbs as inhibitors revealed the test to be specific and reproducible, with an analytical sensitivity of 16 μg per ml of antibody. The reagents remained stable for at least 9 months when kept at 4°C. In the examination of 16 stored sera obtained from 14 patients with proven cases of typhoid fever and 78 serum samples from 75 subjects without typhoid fever, TUBEX was found to be 100% sensitive and 100% specific. The nontyphoid group comprised 26 healthy blood donors, 30 antinuclear antibody (ANA)-negative patients, 9 ANA-positive patients, of whom 1 was positive for anti-DNA antibody, 4 typhus patients, and 6 septicemic patients. In addition, the sera obtained from 11 patients clinically diagnosed as having typhoid fever were all positive in the test. The TUBEX results correlated to some extent, albeit insignificantly (r = 0.38,P = 0.07), with those of an enzyme-linked immunoassay (ELISA) which used a similar detection format (inhibition) and reagents (S. typhi LPS and anti-O9 antibody). TUBEX correlated very well with ELISAs which detected anti-S. typhi LPS IgM (r = 0.58, P = 0.003) or IgG (r = 0.54, P = 0.006) antibodies from the typhoid patients. There was no correlation with the Widal test. The TUBEX test, if performed on slides (instead of tubes) or with soluble antigen (instead of antigen-conjugated magnetic beads), suffered significantly in sensitivity. Direct agglutination tests using LPS-conjugated indicator particles performed either on slides or in microwells also failed to detect antibodies from the majority of typhoid patients. Thus, TUBEX appears to be well designed and well suited for use in the laboratory or by the bedside as a simple, rapid aid to the routine diagnosis of typhoid fever.

Rapid Diagnosis of Typhoid Fever by Enzyme-Linked Immunosorbent Assay Detection of Salmonella Serotype Typhi Antigens in Urine

“…However, surprisingly, the specificity was only 25% when assessed among patients with blood culture-confirmed brucellosis, despite the use of monoclonal antibodies to Vi (an improvement to the ELISA suggested by other investigators). 20 This indicates that Brucella species may carry a Vi-like antigen, or that a Vi-like antigen is released into the urine of patients with bloodstream infections, perhaps as part of the renal component of the acute phase response. We were unable to find previous reports of Vi-like antigen from the urine of patients with brucellosis.…”

“…11 Enzymelinked immunosorbent assay (ELISA) methods have been used to detect antibody to various infectious agents, 12 including Salmonella Typhi. [13][14][15][16][17][18][19][20] However, for typhoid fever, these methods have been hampered by similar limitations of sensitivity and specificity that occur with the use of the Widal agglutination test.…”

Searching for the elusive typhoid diagnostic

“…Additionally, such raised antibody levels frequently cannot be detected in patients with culture confirmed typhoid. Problems have also been encountered during the testing of commercial serological tests, including Typhidot and Tubex [38,39]. These assays were assessed in population-based typhoid surveillance studies in several countries and in all locations the sensitivity and specificity for Tubex and Typhidot was only around 70% and 80% respectively [40,41].…”

Study of TUBEX as a Rapid Diagnostic Test of Typhoid Fever

“…The a-D-Tyvelose is the immunodominant sugar of the O9 determinant. An extremely rare sugar in nature, a-D tyvelose is antigenically different from the b-D-tyvelose found in T. Spiralis or the L-tyvelose in Ascaris lumbricoides [18]. However, the O9 determinant is present not only in S. typhi but also in several other serotypes of Salmonella (serogroup D) such as S. enteritidis and S. sendai.…”

“…However, many of these bacteria are not invasive and may not stimulate a systemic antibody response. The extent to which TUBEX detects infection caused by these salmonellae or the paratyphoid serotypes remains to be investigated [18]. Previously, using the ELISA equivalent of TUBEX we found that serum samples from septicemic patients infected with Salmonella organisms not belonging to serogroup D (one with S. choleraesuis, one with S. johannesburg, and one with S. senftenberg) were negative in the test, whereas that from a patient infected systemically with S. sendai (serogroup D) was weakly positive.…”